Breast cancer is the most frequently diagnosed cancer in American women. It is the leading cause of death in young women under 50 years of age, and is the second most common cause of cancer death among American women. The key to increasing survival is early diagnosis. Early detection through screening mammography saves lives. However, mammography misses as much as 20% of the breast cancer in premenopausal women and 10% in older women. Breast biopsies resulting from abnormal mammograms confirm cancer in only 10-20% of the cases.
While intense effort has been invested in refining the resolution and interpretation of mammography, little has been devoted to the search for tumor markers that may assist in detecting minute amounts of breast cancer. Current serum tumor markers for breast cancer are only useful in cases of widespread disease. (Harris J R, Morrow M, Norton L. Malignant tumors of the breast. In: DeVita V T, Hellman S, Rosenberg S A, eds., In Cancer: principles and practice of oncology. Philadelphia: Lippincott-Raven, (1997).
Recently, there has been increased interest in the possible use of angiogenic factors as tumor markers. In the past few decades, researchers have become increasingly interested in the observation that tumor growth and metastasis are accompanied by significant new blood vessel formation, i.e. angiogenesis. Angiogenic factors have been found associated with several solid tumors such as retinoblastomas and osteosarcomas. Studies have shown that angiogenic factors can be significantly elevated in the serum and urine of breast cancer patients. The levels of certain angiogenic factors have been shown to correlate with the disease stage of the tumor. (Nguyen M., Invest New Drug (1997)).
Approximately 15 angiogenic peptides have been identified and sequenced, including basic fibroblast growth factor (bFGF). (Folkman J, In The molecular basis of cancer, Mendelsohn et al., (eds), W B Saunders, pp 206-232 (1995)). These angiogenic molecules are either released by the tumor cells themselves, or mobilized from extracellular matrix and/or released by host cells, such as macrophages recruited into the tumor.
bFGF, one of the most potent angiogenic factors, has been reported to be widely distributed among normal and neoplastic tissue (Folman J, sura). bFGF is a member of a family of heparin binding growth factors found in a variety of normal and neoplastic tissues. A method for detecting and measuring bFGF using a sandwich immunoassay method is described in U.S. Pat. No. 5,187,062 to Sato et al. A sensitive assay for the detection of bFGF in bodily fluids was not reported until 1991 (Watanabe et al., Biochem. Biophys. Res. Comm. 175:229-235, (1991)), with the first clinical use reported by Fujimoto et al., Biochem. Biophys. Res. Comm. 180:386-392 (1991)). bFGF was elevated in serum of patients with renal cell carcinoma, but was not detected in the urine of these patients. Only 6% of 235 patients with breast cancer had detectable bFGF (>39 pg/ml) using the Watanabe bioassay (Watanabe et al., (Abstract) Molec. Biol. Cell 3S:234a (1992)). An elevated level of bFGF has been found in the urine of patients with a variety of tumors including kidney, bladder, prostate, testicular, breast, colon, lung, brain, ovarian, sarcoma and lymphoma (Nguyen et al., J. Natl. Cancer Inst. 86:356-361 (1994)).
Improvements in the ELISA used for detecting FGF have permitted improved detection of bFGF in urine from subjects with bladder tumors (O'Brien et al., Br. J. Urol. 76:311-314 (1995)), Wilms' tumors (Lin et al., Clin. Cancer Res. 1:327-331 (1995)) and in serum of patients suffering from cervical cancer (Sliutz et al., Cancer Lett. 94:227-231 (1995)). Takei et al. (Clin. Chem. 40:1980-1981 (1994)) measured serum bFGF in patients with breast cancer and found significant elevations in all stages of disease.
The level of bFGF (basic fibroblast growth factor) has been shown to correlate with the disease stage of the tumor. (Nguyen M, Watanabe H, Budson A, Richie J, Hayes D, Folkman J, J Natl Cancer Inst., 86: 356-61 (1994)). However, thus far, use of bFGF in urine or serum samples cannot be used as a screening tool, since there is significant overlap in levels of bFGF between normal subjects and cancer patients. (Nguyen et al., J. Natl. Cancer Inst., supra, and Nguyen M., Invest. New Drug., 15: 29-37 (1997)).
bFGF has also been detected in the cerebrospinal fluid (CSF) of patients with brain tumors but not in controls; the level of bFGF correlated with mitogenic activity in CSF in vitro and with density of microvessels in histological sections (Li et al., Lancet 344:82-86 (1994)).
Angiogenic factors may be useful as markers of therapeutic efficacy and to assess an individual cancer patient's prognosis. Previously elevated urine bFGF levels have been shown to decrease into the normal range following complete surgical removal of tumors. Patients with progressive disease had increased bFGF levels detected after repeat urine samples. (Nguyen et al., J. Natl. Cancer Inst. 86:356-361 (1994)).
Breast cancer arises from the epithelial cells that line the ductal/lobular systems of the milk ducts suggesting that examination of this ductal system or its secretions might reveal signs of early cancer. Breast fluid contains immunoglobulins, proteins, lipids, cholesterol, fatty acids, lactose and hormones including prolactin, growth hormone-like protein, EGF and TGFα, calcitonin and insulin-like growth factor (IGF) (Rose, Cancer Det. Prev., 16:43-51 (1992); and Gann et al., Cancer Epidemiol., Biomarkers & Prev., 6:421-8 (1997)). Breast fluid is typically prevented from escaping from the nipple because the nipple ducts are occluded by constricting bands of smooth muscle, viscous and dried secretions and keratinized epithelium (Petrakis, Epidemiol. Rev. 15:188-195 (1993)).
Patients' nipple fluid (nipple aspirate fluid or “NAF”) has not been extensively investigated as a possible source for breast cancer diagnostic purposes. Factors associated with the success of obtaining NAF include age, (subjects within the age range of 30 to 50 years), subjects having early onset of menarche, subjects of non-Asian race, and subjects with prior lactation. (Wrensch et al., Breast Cancer Res. Treatm. 15:39-51 (1990)).
Prior studies have attempted to detect cancer cells in NAF, but technical difficulties have included a paucity of cancer cells, probably because the cancer obstructs the ducts, and difficulty in distinguishing cancer cells from dyplastic cells. (Wrensch et al., Am. J. Epidemiol., 137:829-33 (1993)). Previous studies with nipple fluid CEA (carcinoembryonic antigen) and PSA (prostate specific antigen) showed significant overlap between the study groups. (Foretova L, Garber J E, Sadowsky N L, Verselis S J, Joseph D M, Andrade A F F, Gudrais P G, Fairclough D, Li F P. Carcinoembryonic antigen in breast nipple aspirate fluid. Cancer Epidemiol Biomark Prevent, 7: 195-8 (1998); and Sauter E R, Daly M, Linahan K, Ehya H, Engstrom P F, Bonney G, Ross E A, Yu H, Diamandis E. Prostate-specific antigen levels in nipple aspirate fluid correlate with breast cancer risk. Cancer Epidemiol Biomark Prevent, 5: 967-70 (1996)).
There remains a need for improved diagnostic methods for breast cancer that are better able to distinguish between normal subjects and those having breast cancer.